Techniques for imaging birefringent objects, such as birefringent molecule imaging (e.g., spindle) in biology and biomedical engineering, require rotational stages operating on inverted microscopes for orienting samples (e.g., biological cells) at a high speed, with a high accuracy, and in an automated manner. Presently, such rotational stages are not commercially available, and there are no known studies/research on this topic in the literature.
Among several model organisms, the mouse is a popular animal widely used in genetic studies and reproductive research. In the injection of mouse embryos and oocytes, the polar body of the embryos must be positioned away from the penetration site to avoid polar body damages and increase the chance of further cellular development. Within the current art for manual associated techniques and technologies, joystick-based and microrobotic mouse embryo injection and embryo orientation is achieved with a holding pipette by repeated vacuum sucking and releasing until the polar body is rotated away from the penetration site. Due to poor controllability, orienting embryos is a slow and trial-error process. Furthermore, the use of a holding pipette makes switching from one embryo to another highly time-consuming. Little or no effort has been spent on speeding up embryo immobilization and orientation control, preventing the realization of full automated, high-speed microrobotic injection.
Embryo orientation may be achieved using a rotational stage mounted on an inverted microscope. In order to observe samples sitting on a rotational stage that is mounted on an inverted microscope, the rotational stage should not introduce any obstruction into the optical path of the inverted microscope. Meanwhile, a sample holder is required to hold the samples on a standard glass slide or Petri dish and importantly, and to keep the sample close enough to the microscope objectives within their working distance. Since the sample is usually not coincident along the rotational axis of the rotational stage, coupled translational motions during rotation cause the target sample to move beyond the field of view. Thus, control methods are needed to keep the sample inside the field of view during sample rotation.
U.S. Pat. Nos. 6,917,420, 6,779,278, 4,891,526, and 6,777,688 are related to rotational stage designs for non-transparent sample imaging. The structures of these stages block the optical path, making them unsuitable for use on inverted microscopes.
U.S. Pat. No. 5,103,338 discloses a rotational stage for positioning objects for microscopic examination. The rotational stage described in this patent does not block the optical path of the microscope. The clamps for holding samples are located on top of the rotating sample holder, which would position a sample outside the small working distances of inverted microscope objectives. The stage is bulky and it is not suited for accommodating and viewing large sample holding devices such as Petri dishes.
The commercial rotational stage offered by Newport Inc. (Irvine, Calif. 92606) which is intended for rotating optical components in optical systems, possesses a through-hole that permit light to pass through. Although such stages do not block the optical path, the clamps for holding samples are located on top of the rotating sample holders, which would position a sample outside the small working distances of inverted microscope objectives. Finally, since stages presented in the above cited prior art were not designed for inverted microscopy use, they cannot be readily mounted on a commonly used motorized XY stage on an inverted microscope.
There arise, however, problems inherent in the use of a rotational stage that is not addressed by the prior art. For example, when the rotational stage rotates through θ° from a fiducial initial rotating position (hereinafter termed a fiducial rotating position), the X- and Y-directional guides also rotate through θ° concomitantly with the stage, which may bring a sample beyond the field of view of the microscope.
In view of the foregoing what is needed is a rotational stage method that overcomes the limitations of the prior art. There is a need for such a rotational stage method that: produces a smooth and fast rotational motion; does not block the light path; possesses a sample clamping mechanism to make the cell sample close enough to the microscope objectives and therefore, within the working distance of the microscope; and has a compact structure to permit the rotational stage to be readily mounted onto a commonly used motorized XY microscopy stage. In addition, control methods for automatic sample orientation control in order to keep the sample inside the field of view while it is rotated by the rotational stage are needed.